The immune regulation and therapeutic potential of the SMAD gene family in breast cancer

Breast cancer is a serious threat to human health. The transforming growth factor-β signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-β signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-β signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.

the formula V = 1/2L*W2.On day 15, mice were euthanized using the cervical dislocation method under anesthesia (0.7% sodium pentobarbital), and tumors were harvested and weighed.Tumor volume (mm 3 ) in each group was graphed over time to monitor tumor growth and assess the synergistic effect of SMAD deletion in vivo.

Immunochemistry analysis
Mouse xenograft tumor tissues were excised and fixed in 10% formalin and paraffin-embedded sections were prepared.For immunochemistry analysis, sections were deparaffinized, hydrated, and boiled for 10 min in 10 mM citrate buffer (pH 6.0), followed by cooling for 20 min at RT. Sequential blocking with 3% BSA was conducted for 1 h to prevent unspecific antibody binding.Staining was performed using corresponding antibodies at 4 °C overnight.Subsequently, a biotinylated secondary antibody was employed.Streptavidin-HRP conjugates and DAB Kit were used as the chromogenic substrate.The sections were then counterstained with haematoxylin.

Drug response analysis
We downloaded and obtained IC50 values for 58 cell lines and 860 drugs from GDSC database (http:// www.cance rrxge ne.org/) 31,32 and analyzed the correlation between SMAD family gene expression and drug IC50 value using the Pearson correlation method.

CCK8 cell viability assay
BT-549 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 10 μg/mL insulin, and 1% penicillin/streptomycin.Cells were incubated in a constant-temperature humidified incubator at 37 °C and 5% CO 2 .For subculture, cells were digested using 0.25% trypsin.BT-549 cells were seeded in 96-well plates with a cell density of 5 × 10 3 cells per well.After incubating overnight, cells were attached and grown in wells.Then, the RPMI 1640 medium was removed and replaced by fresh RPMI-1640 medium containing different concentrations of BAY-1161909 or XK-469.After 24 h of treatment, 10 μL of CCK-8 reagent was added to each well in the dark.Then, the 96-well plate was wrapped with foil and put in the incubator.After 2 h, the absorbance OD values were measured at 450 nm using a microplate reader, and the cell survival rate was calculated according to the following formula.

Apoptosis analysis by flow cytometry
Cells were seeded in six-well plates at a density of 2 × 10 5 cells/well (in triplicate) and grown overnight.Then, cells were treated with different concentrations of BAY-1161909 or XK-469.After 24 h, cells were harvested and then incubated with PI and Annexin V-FITC using an apoptosis detection kit following the manufacturers' instructions.Finally, cells were measured using flow cytometry (Becton Dickinson, FACS Verse, USA).FlowJo7.6 software was used for data analysis.

Ethics statement
We confirmed that all methods are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).Animal experiments were conducted following the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the Ethics Committee of Harbin Medical University and animals were euthanized following the 2020 AVMA Guidelines.The data used in the article is from the TCGA database and is publicly available.

The multi-omics analysis of genes of the SMAD family
We analyzed the expression of genes of the SMAD family in patients with breast cancer from the TCGA database.These genes were significantly dysregulated in cancer tissue (Fig. 1A, Table S1).There was a greater difference in the expression of SMAD2, SMAD3, SMAD4, SMAD5, and SMAD9 between cancer and normal samples.Then, we performed correlation analysis on the expression of genes.Except for the SMAD6 gene, other genes of the SMAD family were positively correlated in patients with breast cancer.In addition, the expression of SMAD3 was positively correlated with that of SMAD6, while SMAD6 expression was negatively correlated with the expression of other genes (Fig. 1B).Log-rank test showed that the expression of genes of the SMAD family could not significantly predict the overall survival (OS) and disease-free survival (DFS) of patients (Figs.S1, S2).

The functional analysis of SMAD family genes
Next, we explored the functions of SMAD family genes (Fig. 2).The SMAD family genes were significantly enriched in cell differentiation, negative regulation of cell proliferation, and regulation of TGF-β receiver signaling pathway.In the pathway enrichment analysis, the SMAD family genes were significantly enriched in the TGF-β signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways, such as gastric cancer and pancreatic cancer.In the TGF-β signaling pathway, TGFβRI, and TGFβRII, ACVR2A, and ACVR2B Cell survival rate% = absorbance OD value of experimental group − absorbance of the blank group ÷ absorbance OD value of the control group − absorbance of the blank group × 100% promoted the expression of SMAD family genes (including SMAD2, SMAD3, and SMAD4), thereby affecting cell cycle and including stem promoter phenotype (Fig. S3).

The expression of SMAD family genes in different stages of breast cancer
We also analyzed the expression of SMAD family genes in different TNM stages of breast cancer (Fig. 3).Except for SMAD1 and SMAD9 genes, other SMAD family genes were significantly and differentially expressed at a certain stage.SMAD3, SMAD6, and SMAD7 were significantly downregulated in stage II (p-value = 0.002 for SMAD3, p-value = 0.041 for SMAD6, and p-value = 0.045 for SMAD7).SMAD4 was significantly downregulated in stage III (p-value = 0.011), and SMAD2 was non-significantly downregulated in stage III (p-value = 0.070).Similarly, we found that the SMAD5 tended to be downregulated in stage IV (p-value = 0.085).These results showed that the SMAD family genes were differentially expressed in different stages of breast cancer.

The correlation between immune cell infiltration and expression of SMAD family genes
Human immune system is deeply involved in the development of breast cancer.Firstly, we used the ESTIMATE algorithm to assess the association of SMAD family genes with immune score, ESTIMATE score, and stromal score (Fig. 4A).We found that the expression of SMAD9 and SMAD6 genes was significantly and positively correlated with immune score, whereas the expression of SMAD2, SMAD3, SMAD4, and SMAD5 was significantly and negatively correlated with immune score (p-value < 0.05).Similarly, the expression of SMAD9 and SMAD7 was significantly and positively correlated with the ESTIMATE score, whereas the expression of SMAD2, SMAD3, SMAD4, and SMAD5 was significantly and negatively correlated with this score (p-value < 0.05).Meanwhile, SMAD1, SMAD6, SMAD7, and SMAD9 were significantly positively correlated with the stromal score (p-value < 0.05).These results indicated that the expression of SMAD2, SMAD3, SMAD4, and SMAD5 was more closely correlated with the immune response.Furthermore, we explored the association between the expression of SMAD family genes and immune cell infiltration scores (Fig. 4B).The expression of SMAS2, SMAD3, SMAD4, and SMAD5 were significantly and positively correlated with the cell infiltration scores of CD8 memory cells and helper T cells, while significantly negatively and correlated with the infiltration scores of CD8 effector cells, regulatory T cells, dendritic cells, natural killer cells, and plasma cells.The expression of SMAS6, SMAD7, and SMAD9 showed the opposite to pattern, and they were significantly and positively correlated with the infiltration of dendritic cells.These results suggest that the SMAD family of genes regulates the immune regulation process of cancer.

The functions of SMAD3 in tumorigenesis
We found that the expression of SMAD3 was correlated with the immune response, including CD8 memory cells and helper T cells.We investigated the effect of SMAD3 knockdown in vivo.SMAD3 knockdown 4T1 cells were subcutaneously injected into mice to construct a xenograft tumor model in mice.SMAD3 knockdown significantly inhibited tumorigenesis (Fig. 5).SMAD3 knockdown suppressed breast cancer growth.The results suggested the synergistic inhibitory effect of SMAD3 knockdown on tumor growth in vivo.Together, these findings demonstrated that SMAD3 knockdown inhibited tumor immune escape in vivo.

The drug sensitivity correlation analysis of SMAD family genes
Finally, we assessed the association between the expression of SMAD family genes and drug sensitivity and heterogeneity (Fig. 6).We downloaded the IC50 values of 860 drugs from the GDSC database.Patients with high expression of SMAD1 showed significantly high sensitivity to bafetinib, rebastinib, MLN-2480, and CEP-32496.Patients with high expression of the SMAD3 exhibited significant resistance to dasatinib and staurosporine, but high sensitivity to tamoxifen, auranofin, BAY-1161909, and XK-469.Patients with high expression of SMAD7 gene exhibited significant resistance to spebrutinib, Bet-BAY-002, AT-13148, VS-5584, and GS-9901.These results indicated that SMAD family genes were associated with anti-cancer drugs.

The effects of SMAD3 on the apoptosis of breast cancer cells
Based on the above results, we found that the expression of SMAD3 was significantly associated with the immune cell infiltration score and the IC50 value of breast cancer drugs.To investigate whether SMAD3 knockdown can synergistically affect the tolerance of breast cancer cells to BAY-1161909 or XK-469, we first investigated the inhibitory effects of individual drugs.The condition of BT-459 cells after SMAD3 knockdown was analyzed by CCK8 and flow cytometry.The results showed that SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, whereas BAY-1161909 and XK-469 increased drug efficacy (Fig. 7A-D).Annexin V/PI staining and flow cytometry analysis were conducted for BT-549 cells 24 h after receiving different doses of BAY-1161909 or XK-469.We also performed Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, which is frequently used to measure apoptosis.SMAD3 knockdown enhanced BAY-1161909-and XK-469-mediated apoptosis (Q2 late apoptosis and Q4 early apoptosis) (Fig. 7E,F).

The genomic variation analysis associated with SMAD3
Based on the above research, we found that the expression of SMAD3 was not only associated with the immune cell infiltration score but was also significantly associated with the IC50 value of breast cancer drugs.Based on SMAD3 gene expression, patients with breast cancer were divided into the high-expression group and the lowexpression group (Fig. 8).To analyze the effect of SMAD3 gene expression on patients' genomes, we explored the gene mutation distribution profiles of the two groups.Mutations in the TP53, PIK3CA, TTN, CDH1, GATA3, KMT2C, MUC16, SYNE1, PTEN, and MAP3K1 genes were present in at least 7% of patients with low expression of SMAD3, while mutations in the PIK3CA, TP53, TTN, CDH1, GATA3, MUC16, MAP3K1, and KMT2C genes were present in at least 10% of patients with high expression of SMAD3.In summary, there were differences in frequent gene mutations between the high-and low-expression groups, but most gene mutations were similar.In addition, we analyzed the relationship between SMAD3 gene expression and tumor mutation load.SMAD3 gene expression was not related to tumor mutation load in patients with breast cancer, which also verifies the results of gene mutation distribution map analysis.

Discussion
In this study, we used the multi-omics data of patients with breast cancer from TCGA for analysis.Except for SMAD6, the expression of other SMAD family genes was positively correlated.We also found that the SMAD family genes were significantly enriched in cancer-related pathways.In addition, the expression of SMAD family genes was significantly correlated with immune cell infiltration scores.We demonstrated that SMAD3 knockdown inhibited tumor immune escape in vivo.We also analyzed the association between the SMAD family genes and the IC50 value of drugs.Patients with high expression of SMAD3 showed significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin.Furthermore, we analyzed the association between the expression of SMAD3 and anesthetic drugs.In addition, we confirmed that SMAD3 knockdown had www.nature.com/scientificreports/synergistic effects on BAY-1161909-and XK-469-induced apoptosis of breast cancer cells.Our results indicated that SMAD family genes play an important role in breast cancer.We found a significant negative correlation between SMAD3 expression and immune score or ESTIMATE score.Moreover, SMAD3 expression was significantly and positively correlated with the infiltration scores of CD8 memory cells and helper T cells while negatively correlated with the infiltration scores of CD8 effector cells, regulatory T cells, dendritic cells, natural killer cells, and plasma cells.Chung et al. found that SMAD3 activation in neutrophil TANs was associated with increased N2 phenotype and poor prognosis of NSCLC, while SMAD3 inhibition promoted the polarization of TANs to the anti-tumor N1 phenotype, thereby inhibiting the development of lung cancer 33 .TGF-β enhanced PD-1 expression in an SMAD3-dependent manner.Therefore, TGF-β regulates the expression of several genes to reduce CTL differentiation and function in cytotoxic T cells 34,35 .However, in our study, SMAD3 knockdown suppressed breast cancer growth, which demonstrated that SMAD3 knockdown can inhibit tumor immune escape in vivo.
At the same time, we found that SMAD4 similarly affects the immune.The TGF-β induced SMAD3/SMAD4 complex-mediated activation of activated PKA to trigger carboxyl terminal SRC kinase (CSK)-mediated inhibition of proximal TCR signal and prevent unexpected T cell activation.The expression of TGFBR1 in NK cells can enhance cancer cell metastasis and accelerate the growth of fibrosarcoma, indicating that TGF-β activates SMAD4 to inhibit NK cell-mediated monitoring in cancer metastasis 36 .
Endocrine therapy for breast cancer has a history of more than 100 years and plays an indispensable role in the treatment of HR-positive patients.Tamoxifen is the most widely used drug for endocrine therapy, which mainly competitively binds to tumor cell ER, thereby preventing the promoting effect of estrogen on tumor cell growth and proliferation [37][38][39] .We found that patients with high expression of SMAD3 showed significantly high sensitivity to tamoxifen and auranofin.Prahlad et al. found that auranofin, an FDA-approved thioredoxin reductase inhibitor, caused specific cell death and impaired the growth of TNBC cells 40 .Joo-Eun et al. found that mesupron combined with auranofin may exhibit synergistic anti-cancer effects 41 .There are also studies indicating that combined treatment with auranofin and trametinib synergistically induces apoptosis in breast cancer cells 42 .In addition, we also found that patients with high expression of SMAD3 showed significant resistance to dasatinib and staurosporine.Single-agent dasatinib has limited activity in patients with triple-negative breast cancer 43 .We also found that SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, whereas BAY-1161909 and XK-469 increased drug efficacy.Furthermore, SMAD3 knockdown enhanced BAY-1161909-and XK-469-induced apoptosis.The synergy between dasatinib and doxorubicin warranted the re-assessment of dasatinib as an effective agent in multi-drug regimens for treating invasive breast cancers 44 .

Conclusion
We used multi-omics data of breast cancer patients from TCGA to explore the functions of SMAD family genes.We found that the expression of other SMAD family genes was positively correlated.The SMAD family genes were significantly enriched in cancer-related pathways.In addition, the expression of SMAD family genes was significantly correlated with immune cell infiltration scores.Furthermore, the high expression of SMAD3 was associated with significant resistance to dasatinib and staurosporine while high sensitivity to tamoxifen and auranofin.In addition, we explored the genomic characteristics of SMAD3.Our results indicated that SMAD family genes play an important role in breast cancer.

Figure 1 .
Figure 1.The multi-omics analysis of genes of the SMAD family.(A) The differential expression of SMAD family genes in breast cancer samples and normal samples.(B) The expression correlation of these genes.(C) The genomic variations of SMAD family genes.

Figure 2 .Figure 3 .
Figure 2. The functional enrichment analysis of genes of the SMAD family: (A) biological process of gene ontology, (B) molecular function of gene ontology, (C) cellular component of gene ontology, and (D) Kyoto Encylopaedia of Genes and Genomes pathways.

Figure 4 .
Figure 4.The correlation between the expression of genes of the SMAD family and immune cell infiltration score.(A) The scores of the immune score, stromal score, and estimate score.(B) The immune cell infiltration score in breast cancer samples.

Figure 6 .
Figure 6.The drug sensitivity correlation analysis of SMAD family genes.(A) The scatter plot.(B) The box plot.